Crystal structure of the in-cell Cry1Aa purified from Bacillus thuringiensis.

In-cell protein crystals which spontaneously crystallize in living cells, have recently been analyzed in investigations of their structures and biological functions. The crystals have been challenging to analyze structurally because of their small size. Therefore, the number of in-cell protein crystals in which the native structure has been determined is limited because most of the structures of in-cell crystals have been determined by recrystallization after dissolution. Some proteins have been reported to form intermolecular disulfide bonds in natural protein crystals that stabilize the crystals. Here, we focus on Cry1Aa, a cysteine-rich protein that crystallizes in Bacillus thuringiensis (Bt) and forms disulfide bonds. Previously, the full-length structure of 135 kDa Cry1Ac, which is the same size as Cry1Aa, was determined by recrystallization of dissolved protein from crystals purified from Bt cells. However, the formation of disulfide bonds has not been investigated because it was necessary to replace cysteine residues to prevent aggregation of the soluble protein. In this work, we succeeded in direct X-ray crystallographic analysis using crystals purified from Bt cells and characterized the cross-linked network of disulfide bonds within Cry1Aa crystals. [Display omitted] • The full-length structure of Cry1Aa crystallized in Bt cells was determined. • The intermolecular disulfide bonds between Cys820-Cys1023 form cross-linked networks within crystals. • This work will give insights into the mechanism of Cry1Aa crystal dissolution in the insect midgut. [ABSTRACT FROM AUTHOR]